ABSTRACT Treatment of acute ischemic stroke with tPA can significantly improve neurological outcomes. However, many patients do not recanalize with one recent study showing that only 53% of patients receiving IV tPA were recanalized at 24 hours. And while endovascular therapy can significantly increase reperfusion rates in patients with large vessel disease, not all occlusions are accessible for endovascular treatment and many community hospitals are not able to perform these procedures. Thus, for many patients thrombolysis remains the only treatment option. In addition to the limited efficacy of tPA, thrombolysis carries a small but significant risk of symptomatic intracerebral hemorrhage (sICH), increasing from ~0.5% in untreated patients to up to 5% in some studies. Importantly, the risk of fatal ICH with thrombolysis rises to ~2% in patients treated with the current recommended dose of tPA. Studies with lower doses of tPA show a reduction in sICH however these studies have not demonstrated noninferiority of the lower dose compared to standard therapy. In contrast to stroke, dosing recommendations of thrombolytic tPA for myocardial infarction or pulmonary embolism can be nearly 2-fold higher than the dose for ischemic stroke, suggesting that the balance between safety and efficacy in stroke may limit the potential efficacy of tPA. Thus, understanding the molecular pathways triggered by thrombolytic tPA that increase the incidence of hemorrhagic conversion could profoundly improve the treatment of ischemic stroke. By targeting these pathways and reducing the risk of sICH it should be possible to not only make thrombolysis safer but also to make it more effective by permitting increased doses of tPA to be used to promote reperfusion in more patients. In earlier work, we established an association between tPA?mediated cell signaling in the neurovascular unit (NVU) and blood brain barrier (BBB) regulation. Acting through tPA cleavage and activation of platelet- derived growth factor C (PDGF-C) and binding of active PDGF-C to the PDGF receptor ? (PDGFR?) on perivascular astrocytes, this pathways induces increased vascular permeability and thrombolysis associated hemorrhage. In the present application we present data demonstrating that tPA mediated PDGFR? signaling in perivascular astrocytes enhances the activity of VEGF-A in the neurovascular unit leading to BBB breakdown through disruption of endothelial cell tight junction complexes. This hypothesis is based upon strong preliminary data linking tPA to the phosphorylation of the tight junction protein occludin at S490 and to increased BBB permeability. This activity is dependent on PKC?, and inhibition PKC? or expression of a non- phosphorylatable occludin mutant, significantly reduces thrombolytic tPA-associated ICH in a murine model of thrombotic stroke with thrombolysis.